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The Effect of Dimercaprol on Amine Oxidase and Amino Acid Decarboxylase of the Normotensive and Hypertensive Rat Kidney*
JOURNAL OP
844
THE
AMERICAN PHARMACEUTICAL ASSOCIATION
centrifuge a t 2,500 r.p.m. for one minute and decant the upper layer into a low-actinic 250-ml. suction flask. Repeat this extraction two times more. Evaporate the petroleum ether extract on a steam bath using a stream of nitrogen and partial vacuum. Take up the residue in about 50 ml. of petroleum ether, add 5 Gm. of Florex XXS and continue with Method I, starting with, “. . . fit with an air condenser . . .” Calculate as in Method I. Results obtained with this method on various preparations are listed in Table I. Comments.-This method furnishes the sharpest separations of vitamin A and its degradation products (yieldiiig a blue color) from vitamin D, although it did not eliminate the yellow off-color, encountered in certain preparations. I n order t o increase the precision of this procedure, a 1.00-ml. aliquot of A & D powder (sample No. 13, Table I) was chromatographed instead of an 0.5-ml. aliquot, as described in the procedurc. Although some difliculty was encountered in absorbing the stationary phase, the results were satisfactory.
CONCLUSIONS A basic method and three modifications for the chemical determination of vitamin D in presence of vitamin .4 are presented. We prefer t o apply first the basic method (Method I ) to a sample of unknown composition. If, as mentioned under “Comments” in Method I, a yellow off-color is encountered in the colorimetric step, Method I1 would be used. If for any reason the vitamin D cannot be extracted directly (as per Method 11). Method I11 would be substituted. I t has the advantage of a low “blue” blank, but is more time-consuming than Method 11.
Vol. XLVII. No. 12
Method I V may be useful for special situations in which the ratio of vitamin A t o vitamin D is considerably greater than 10: 1. In the case of low potency products (i. e., less than 1,000 units D per dosage unit) Method I1 is preferred. The size of the sample and the dilution steps are adjusted, so that the final solution in the colorimetric step contains about 400 units vitamin D when measured in a 1-ctn. cell. The sensitivity may be increased further, t o measure as low as 200 units D, by use of a 2-cm. cell. All procedures are relatively rapid. The basic method and Method I1 require less than 8 hours working time to run duplicate samples and standards. Method I11 requires one additional hour, and Method I V , a total of about 11 hours working time.
REFERENCES ( 1 ) ‘l‘scliapke, H., aiid Plessing, H , Die Phuutrresir. 12, 2tli2(lY57). (2) Tschapke. H., and Plessinp, H . , I?ttcr?L. %. V i l a r n i s I
(10) Wiss, O . , and Gloor, U . , Hoppe-Seyfer’s Z. physiol. Chem., 310, 263(1958). (11) Gloor, U.. private communication. (12) Nield. C. H., Russel, W. C., and Zimmerli, A,, J . B i d . C h e m . , 136, 73(1940). (13) Mulder, F. J . , Rohorgh, J. R . , DeMan. T h . J., Kenning, K. J., and Hanewald, K. H., Rec. Irau. chim., 76, 733(1957). (1.4) Napoli, J . , and Senkowski, B., Manuscript in preparation for publication.
The Effect of Dimercaprol on Amine Oxidase and Amino Acid Decarboxylase of the Normotensive and Hwertensive Rat Kidnev* J
I
J
By DUANE G. WENZEL and GEORGE N . BECKLOFF T h e administration of dimercaprol to normotensive and hypertensive rats produced pressor and antihypertensive effects, respectively. Kidney amino acid decarboxylase and amine oxidase activities were determined manometrically using ( +)-3,4-dihydroxyphenylalanine (DOPA) as a substrate. Decarboxylase/amine oxidase ratios are presented as suggestive evidence that the dimercaprol-induced changes in blood pressure are related to amino acid metabolism. sulfhydryl compounds are true antihypertensives as they lower only elevated blood pressures. Normal pressures are raised (1). A possible explanation for this activity can ANY
* Received April 25, 1958, from the School of Pharmacy, University of Kansas, Lawrence. Abstracted from a portion of the data contained in a thesis submitted to the Graduate School of the University of Kansas by George N . Beckloff in partial fulfillment of the requirements of the degree of Master of Science. Supported b y a grant from the University of Kansas General Research Fund.
he related to t h e hypothesis t h a t liyl~ertensioii results from the improper metabolism of amino acids and t h a t t h e sulfhydryl compounds correet this metabolic fault. T h e basis of this hypothetical metabolic fault would he a relative increase in the rate of amino acid decarhoxylatiori compared to t h e rate of deamination. This implies t h a t the pressor agent is a n amine. There is considerable evidence t o support t h e possible
1 )cccnihrr 19.58
SCIENTIFIC EDITION
role of pressor amines in hypertension, and some equally contradictory information. Supporting evidence includes the fact that in renal anoxia, a popular method for t h e production of experimental hypertension, t h e amine oxidase is adversely affected as it is aerobic, while the process of amino acid decarboxylation is anaerobic and would be unaffected. It has been observed that a decreased oxygen consumption occurs with the ischemic kidney (2). Holtz, et al. (3, 4), have demonstrated with anaerobic kidney tissue t h a t (- ) -3, 4-dihydroxypenylalanine (DOPA) is converted into t h e pressor hydroxytyramine. I n t h e presence of adequate oxygen this pressor agent was converted t o the depressor dihydroxyphenylacetaldehyde. Similar results were obtained by Bing ( 5 , 6) who obtained a pressor substance bv perfusing a n ischemic c a t kidney with DOPA. Extending this observation t o humans, Oster and Sorkin (7) reported the injection of DOPA t o produce stronger pressor reactions with longer durations i n hypertensive individuals than in normotensives. T h e blood of renal-hypertensive dogs demonstrates a positive tyramine test (8) although some (9) have been unable to obtain this reaction. Blood and urine total amine levels tend to be raised in hypertension (10). It may be significant t h a t both these values in malign a n t hypertension are close t o normal. Page and Reed (11) have presented evidence to deny the role of improper amino acid metabolism as a causative factor in, at least, experimental hypertension. They found DOPA to produce a pressor response in normal, hypertensive, hypophysectomized, and nephrectomized rats. I n fact, t h e rise was greatest in the nephrectomized animals. T h e hypothesis tested in this communication was that the characteristic effects of dimercaprol o n blood pressure are related to alterations in the ratio of kidney decarboxylasejamine oxidase activities.
EXPERIMENTAL. Methods.-Male, black-hooded rats weighing approximately 200 Gm. were used as experimental animals for the production of renal hypertension. The procedure of Drury (12) was used to induce hypertension. Animals were anesthetized by the intraperitoneal administration of 35 mg./Kg. sodium pentobarbital and the kidneys exposed by a midline abdominal incision. A nichrome wire (0.0145 inch 0. D.)was placed along the axis of the 1 Grateful acknowledgment is made to Dr. J . H. Tilden, Rli 1,illy nncl C o m p a n y , f o r essential details of this prrrredure.
84.5
right renal artery and the artery and wire tied snugly together with a nylon thread using two sets of square knots. If the knot was tied with the correct amount of tension, a slight resistance was encountered to the removal of the wire, but the kidney quickly reverted to its normal color. The left kidney was then removed using care to avoid injury to the adrenal gland. Blond pressure readings were obtained from the tails of unanesthetized animals with the microphonic manometer described by Friedman and Freed (13). Normal systolic pressures averaged about 100 mm. Hg. One to three weeks after constriction of the renal artery the pressure reached about 150 mm. Hg. The mortality rate was approximately 4570. Four groups of ten rats per group were established to determine the kidney decarboxylase and amine oxidase activities. Two groups were normotensive and two hypertensive. One normal group and one hypertensive group were treated with dimercaprol prior to the kidney enzyme determinations. The dimercaprol in oil (40 mg./Kg.) was injected intraperitoneally and the blood pressure determined a t rapid intervals until it reached a level where it became stabilized. A t this point the animal was quickly sacrificed by a blow on the head, the remaining kidney removed and placed in a saline bath a t a temperature of 3'. Two animals were treated in this manner in quick succession and the kidneys pooled for further treatment and study. Enzyme extracts of the kidneys were prepared in a cold room (3 "). The kidneys were first trimmed of fascia and necrotic tissue and gently pressed between filter paper t o remove excessive moisture. They were then weighed and ground with 1.5 Gm. of purified sand for ten minutes. Three milliliters of M/15 phosphate buffer, p H 6.80, were added to the mortar and the grinding continued for ten more minutes. The mixture was transferred with washing to a centrifuge tube and the volume adjusted with buffer to correspond t o a concentration of 7.2 ml. per Gm. of tissue. The mixture was shaken for one hour with a mechanical shaker, centrifuged at low speed for five minutes, and the supernatant liquid carefully decanted from the precipitate and saved. Enzyme activities were determined manometrically using ( A ) -3,4-dihydroxyphenylalanine(DOPA) as the substrate. Amine oxidase activity was determined by the oxygen uptake according to the procedure of Raska (2). Decarboxylase was measured by the COz evolution (14) on separate samples. Carbon dioxide evolution or oxygen uptake was measured for two hours and the results divided by two t o give the activity per hour. Two or three replications were obtained from each pooled kidney pair, and the mean values used. In those instances where only two determinations were made, the weight of the combined kidneys was inadequate to prepare enough enzyme extract for six determinations. The mean C O Z / O ~per hour ratios were calculated for each experimental group. As the kidneys from two animals were pooled to obtain the COl/Oz values and there were ten animals per group, the mean values represent a total of five individual ratios. The standard error of the means and the significance of the differences o f the means were c:~lculnted ( I 5).
846
JOURNAL OF THE
TABLE I.-EPFECT
OF
AMERICAN PHARMACEUTICAL ASSOCIATION Vol. XLVII, No. 12
DIMERCAPROL ON BLOOD PRESSURES AND KIDNEYENZYMES OP NORMOTENSIVE A N D HYPERTENSIVE RATS
Normotensive
Mean blood pressures (mm. .Hg) After Dimercaprol Mean COZliberation in pl./hr. Mean OZuptake in pl,/hr. Mean C O ~ / O ~ r a t i o s ~
Controlsa
99.5 f 1.91
....
31 (7-42) 268 ( 193-312) 0.115 f 0.005 ( .098-. 129)
~
Hypertensive
149 f 1.82 .... 41 (12-56) 297 (220-353) 0.134 =t0.021 ( ,051-0.239)
Dimercaprol-Treated’ Normotensive Hypertensive
100.6 f 2.47 105.4 f 2.45 40 (24-58) 247 (200-332) 0.165 f 0.021 ,208-y.
~
-
147.8 f 1.82 123.0 f 2.23 29 (10-42) 298 (248-351) 0.098 f 0.018 127’ .05
\ y2 \A
NS
P<0.05
b
-
Figures in parentheses are the ranges. COt/Oz ratios are the means of the individual ratios.
RESULTS AND DISCUSSION
SUMMARY
The effects of dimercaprol on the blood pressures of the normotensive and hypertensive rats were essentially the same a s reported for humans (6). Dimercaprol significantly lowered the blood pressure of the hypertensive animals and, although the increase in pressure of the normotensive animals was too little to be significant, all normal animals responded quite uniformly. The effects of hypertension and dimercaprol on DOPA decarboxylase and amine oxidase are somewhat equivocal. If the individual C02/02 ratios of Table I are compared without regard for significance, i t is seen that both hypertension and the effect of dimercaprol on normotensive animals increase the ratios. The implication is that renal anoxia and dimercaprol raise the blood pressure through the same mechanism; namely, the increased production of pressor amines. The difference in the ratios of the normotensive controls and the normotensive-treated animals and the difference between the normotensive-treated and the hypertensive-treated are significant, but barely so a t the 5% level (14). The mean values for amine oxidase activities are quite close considering the ranges involved. Cruz Coke, et al. (16), have reported no difference in the oxygen consumption of normal and ischemic rat kidneys, in their case, however, without a substrate. The greatest apparent change observed in the Dresent study was in the DOPA decarboxylase activity. If the effects on the ratios and presumably on the pressor action are real, they appear to be related to the relative increase in decarboxylase activity. The antihypertensive action of dimercaprol may be similar to the action of hydralazine which reduces DOPA-decarboxylase activity (17). Both will chelate trace metals required for enzymatic activity.
1. Dimercaprol was administered to normotensive and hypertensive rats and their kidney enzyme extracts tested for amino acid decarboxylase and oxidase activities using DOPA as a substrate. Untreated normotensive and hypertensive controls were similarly tested. 2. Although all results were not significant, t h e evidence was suggestive t h a t both the antihypertensive effect of dimercaprol for hypertensive animals a n d i t s pressor action in normotensive animals are related to amino acid metabolism.
REFERENCES (1) Schroeder, H. A,. J. Clin. Invest., 30, 672(1951). (2) Raska, S. B., J . Ezpll. Med., 78,75(1943). (3) Holtz. P.. Heise, R., and Liidtke, K.. Arch. cxpfl. Path. Pharmakol.. 191, 87(1939). (4) Holtz, P., Credner, K., and Koepp, W.. ibid., 200, 356(1942). (5) B‘ing, R. J., A m . J . Physiol., 132,497(1941). 22.5(1041) (6) Bing, R. J., and Zucker, M. B., J . E r p f l . Med., 74. ..,. (7) Oster, K. A,, and Sorkin. S. Z., Proc. SOL.Exptl. Biol. Med., 51, 67(1942). (8) Wolf, H. J., and Heinsen, H. A., Arch. expfl. Pafhol. Pharmakol.. 179, 15(1935). (9) Govaerts, P.. Bull. acad. roy. mLd. B d g . , 4, 357(1939). (10) Schryder, H. A., “Hypertensive Diseases, Causes and Control, Lea and Fehiger, Philadelphia, 1953, p. 133. (11) Page, E. W.. and Reed, R., A m . J . Physiol., 143, ___\.I
__,.
133(194.5~ ___,_”
(12) Drury, D. R., J . Expll. Med., 68, 693(1938). (13) Friedman, M.. and Freed, S. C., Proc. SOC. Exfd. Biol. Med., 70, 670(1949). (14) Schales, O., in “Methods in Enzymology,” Vol. 11. Colowick, S. P., and Kaplan, N. O., Editors, Academic Press Inc., New York. 1855, p. 195. (15) Trelease. S. F., “The Scientific Paper,” 2nd ed., The Williams and Wilkins Company, Baltimore, 1951, p. 26. (16),Cruz Coke, E., Niemeyer, H., and FernandezPo elaire J. Bol. SOC. b i d . s p n . 2, 15(1944). 8 7 ) Pi&, H. M., Jr.. Te;tlebaum, S.. and Schwarti, P. L., Federalion Proc., 14, Part 1, 113(1955).
844
THE
AMERICAN PHARMACEUTICAL ASSOCIATION
centrifuge a t 2,500 r.p.m. for one minute and decant the upper layer into a low-actinic 250-ml. suction flask. Repeat this extraction two times more. Evaporate the petroleum ether extract on a steam bath using a stream of nitrogen and partial vacuum. Take up the residue in about 50 ml. of petroleum ether, add 5 Gm. of Florex XXS and continue with Method I, starting with, “. . . fit with an air condenser . . .” Calculate as in Method I. Results obtained with this method on various preparations are listed in Table I. Comments.-This method furnishes the sharpest separations of vitamin A and its degradation products (yieldiiig a blue color) from vitamin D, although it did not eliminate the yellow off-color, encountered in certain preparations. I n order t o increase the precision of this procedure, a 1.00-ml. aliquot of A & D powder (sample No. 13, Table I) was chromatographed instead of an 0.5-ml. aliquot, as described in the procedurc. Although some difliculty was encountered in absorbing the stationary phase, the results were satisfactory.
CONCLUSIONS A basic method and three modifications for the chemical determination of vitamin D in presence of vitamin .4 are presented. We prefer t o apply first the basic method (Method I ) to a sample of unknown composition. If, as mentioned under “Comments” in Method I, a yellow off-color is encountered in the colorimetric step, Method I1 would be used. If for any reason the vitamin D cannot be extracted directly (as per Method 11). Method I11 would be substituted. I t has the advantage of a low “blue” blank, but is more time-consuming than Method 11.
Vol. XLVII. No. 12
Method I V may be useful for special situations in which the ratio of vitamin A t o vitamin D is considerably greater than 10: 1. In the case of low potency products (i. e., less than 1,000 units D per dosage unit) Method I1 is preferred. The size of the sample and the dilution steps are adjusted, so that the final solution in the colorimetric step contains about 400 units vitamin D when measured in a 1-ctn. cell. The sensitivity may be increased further, t o measure as low as 200 units D, by use of a 2-cm. cell. All procedures are relatively rapid. The basic method and Method I1 require less than 8 hours working time to run duplicate samples and standards. Method I11 requires one additional hour, and Method I V , a total of about 11 hours working time.
REFERENCES ( 1 ) ‘l‘scliapke, H., aiid Plessing, H , Die Phuutrresir. 12, 2tli2(lY57). (2) Tschapke. H., and Plessinp, H . , I?ttcr?L. %. V i l a r n i s I
(10) Wiss, O . , and Gloor, U . , Hoppe-Seyfer’s Z. physiol. Chem., 310, 263(1958). (11) Gloor, U.. private communication. (12) Nield. C. H., Russel, W. C., and Zimmerli, A,, J . B i d . C h e m . , 136, 73(1940). (13) Mulder, F. J . , Rohorgh, J. R . , DeMan. T h . J., Kenning, K. J., and Hanewald, K. H., Rec. Irau. chim., 76, 733(1957). (1.4) Napoli, J . , and Senkowski, B., Manuscript in preparation for publication.
The Effect of Dimercaprol on Amine Oxidase and Amino Acid Decarboxylase of the Normotensive and Hwertensive Rat Kidnev* J
I
J
By DUANE G. WENZEL and GEORGE N . BECKLOFF T h e administration of dimercaprol to normotensive and hypertensive rats produced pressor and antihypertensive effects, respectively. Kidney amino acid decarboxylase and amine oxidase activities were determined manometrically using ( +)-3,4-dihydroxyphenylalanine (DOPA) as a substrate. Decarboxylase/amine oxidase ratios are presented as suggestive evidence that the dimercaprol-induced changes in blood pressure are related to amino acid metabolism. sulfhydryl compounds are true antihypertensives as they lower only elevated blood pressures. Normal pressures are raised (1). A possible explanation for this activity can ANY
* Received April 25, 1958, from the School of Pharmacy, University of Kansas, Lawrence. Abstracted from a portion of the data contained in a thesis submitted to the Graduate School of the University of Kansas by George N . Beckloff in partial fulfillment of the requirements of the degree of Master of Science. Supported b y a grant from the University of Kansas General Research Fund.
he related to t h e hypothesis t h a t liyl~ertensioii results from the improper metabolism of amino acids and t h a t t h e sulfhydryl compounds correet this metabolic fault. T h e basis of this hypothetical metabolic fault would he a relative increase in the rate of amino acid decarhoxylatiori compared to t h e rate of deamination. This implies t h a t the pressor agent is a n amine. There is considerable evidence t o support t h e possible
1 )cccnihrr 19.58
SCIENTIFIC EDITION
role of pressor amines in hypertension, and some equally contradictory information. Supporting evidence includes the fact that in renal anoxia, a popular method for t h e production of experimental hypertension, t h e amine oxidase is adversely affected as it is aerobic, while the process of amino acid decarboxylation is anaerobic and would be unaffected. It has been observed that a decreased oxygen consumption occurs with the ischemic kidney (2). Holtz, et al. (3, 4), have demonstrated with anaerobic kidney tissue t h a t (- ) -3, 4-dihydroxypenylalanine (DOPA) is converted into t h e pressor hydroxytyramine. I n t h e presence of adequate oxygen this pressor agent was converted t o the depressor dihydroxyphenylacetaldehyde. Similar results were obtained by Bing ( 5 , 6) who obtained a pressor substance bv perfusing a n ischemic c a t kidney with DOPA. Extending this observation t o humans, Oster and Sorkin (7) reported the injection of DOPA t o produce stronger pressor reactions with longer durations i n hypertensive individuals than in normotensives. T h e blood of renal-hypertensive dogs demonstrates a positive tyramine test (8) although some (9) have been unable to obtain this reaction. Blood and urine total amine levels tend to be raised in hypertension (10). It may be significant t h a t both these values in malign a n t hypertension are close t o normal. Page and Reed (11) have presented evidence to deny the role of improper amino acid metabolism as a causative factor in, at least, experimental hypertension. They found DOPA to produce a pressor response in normal, hypertensive, hypophysectomized, and nephrectomized rats. I n fact, t h e rise was greatest in the nephrectomized animals. T h e hypothesis tested in this communication was that the characteristic effects of dimercaprol o n blood pressure are related to alterations in the ratio of kidney decarboxylasejamine oxidase activities.
EXPERIMENTAL. Methods.-Male, black-hooded rats weighing approximately 200 Gm. were used as experimental animals for the production of renal hypertension. The procedure of Drury (12) was used to induce hypertension. Animals were anesthetized by the intraperitoneal administration of 35 mg./Kg. sodium pentobarbital and the kidneys exposed by a midline abdominal incision. A nichrome wire (0.0145 inch 0. D.)was placed along the axis of the 1 Grateful acknowledgment is made to Dr. J . H. Tilden, Rli 1,illy nncl C o m p a n y , f o r essential details of this prrrredure.
84.5
right renal artery and the artery and wire tied snugly together with a nylon thread using two sets of square knots. If the knot was tied with the correct amount of tension, a slight resistance was encountered to the removal of the wire, but the kidney quickly reverted to its normal color. The left kidney was then removed using care to avoid injury to the adrenal gland. Blond pressure readings were obtained from the tails of unanesthetized animals with the microphonic manometer described by Friedman and Freed (13). Normal systolic pressures averaged about 100 mm. Hg. One to three weeks after constriction of the renal artery the pressure reached about 150 mm. Hg. The mortality rate was approximately 4570. Four groups of ten rats per group were established to determine the kidney decarboxylase and amine oxidase activities. Two groups were normotensive and two hypertensive. One normal group and one hypertensive group were treated with dimercaprol prior to the kidney enzyme determinations. The dimercaprol in oil (40 mg./Kg.) was injected intraperitoneally and the blood pressure determined a t rapid intervals until it reached a level where it became stabilized. A t this point the animal was quickly sacrificed by a blow on the head, the remaining kidney removed and placed in a saline bath a t a temperature of 3'. Two animals were treated in this manner in quick succession and the kidneys pooled for further treatment and study. Enzyme extracts of the kidneys were prepared in a cold room (3 "). The kidneys were first trimmed of fascia and necrotic tissue and gently pressed between filter paper t o remove excessive moisture. They were then weighed and ground with 1.5 Gm. of purified sand for ten minutes. Three milliliters of M/15 phosphate buffer, p H 6.80, were added to the mortar and the grinding continued for ten more minutes. The mixture was transferred with washing to a centrifuge tube and the volume adjusted with buffer to correspond t o a concentration of 7.2 ml. per Gm. of tissue. The mixture was shaken for one hour with a mechanical shaker, centrifuged at low speed for five minutes, and the supernatant liquid carefully decanted from the precipitate and saved. Enzyme activities were determined manometrically using ( A ) -3,4-dihydroxyphenylalanine(DOPA) as the substrate. Amine oxidase activity was determined by the oxygen uptake according to the procedure of Raska (2). Decarboxylase was measured by the COz evolution (14) on separate samples. Carbon dioxide evolution or oxygen uptake was measured for two hours and the results divided by two t o give the activity per hour. Two or three replications were obtained from each pooled kidney pair, and the mean values used. In those instances where only two determinations were made, the weight of the combined kidneys was inadequate to prepare enough enzyme extract for six determinations. The mean C O Z / O ~per hour ratios were calculated for each experimental group. As the kidneys from two animals were pooled to obtain the COl/Oz values and there were ten animals per group, the mean values represent a total of five individual ratios. The standard error of the means and the significance of the differences o f the means were c:~lculnted ( I 5).
846
JOURNAL OF THE
TABLE I.-EPFECT
OF
AMERICAN PHARMACEUTICAL ASSOCIATION Vol. XLVII, No. 12
DIMERCAPROL ON BLOOD PRESSURES AND KIDNEYENZYMES OP NORMOTENSIVE A N D HYPERTENSIVE RATS
Normotensive
Mean blood pressures (mm. .Hg) After Dimercaprol Mean COZliberation in pl./hr. Mean OZuptake in pl,/hr. Mean C O ~ / O ~ r a t i o s ~
Controlsa
99.5 f 1.91
....
31 (7-42) 268 ( 193-312) 0.115 f 0.005 ( .098-. 129)
~
Hypertensive
149 f 1.82 .... 41 (12-56) 297 (220-353) 0.134 =t0.021 ( ,051-0.239)
Dimercaprol-Treated’ Normotensive Hypertensive
100.6 f 2.47 105.4 f 2.45 40 (24-58) 247 (200-332) 0.165 f 0.021 ,208-y.
~
-
147.8 f 1.82 123.0 f 2.23 29 (10-42) 298 (248-351) 0.098 f 0.018 127’ .05
\ y2 \A
NS
P<0.05
b
-
Figures in parentheses are the ranges. COt/Oz ratios are the means of the individual ratios.
RESULTS AND DISCUSSION
SUMMARY
The effects of dimercaprol on the blood pressures of the normotensive and hypertensive rats were essentially the same a s reported for humans (6). Dimercaprol significantly lowered the blood pressure of the hypertensive animals and, although the increase in pressure of the normotensive animals was too little to be significant, all normal animals responded quite uniformly. The effects of hypertension and dimercaprol on DOPA decarboxylase and amine oxidase are somewhat equivocal. If the individual C02/02 ratios of Table I are compared without regard for significance, i t is seen that both hypertension and the effect of dimercaprol on normotensive animals increase the ratios. The implication is that renal anoxia and dimercaprol raise the blood pressure through the same mechanism; namely, the increased production of pressor amines. The difference in the ratios of the normotensive controls and the normotensive-treated animals and the difference between the normotensive-treated and the hypertensive-treated are significant, but barely so a t the 5% level (14). The mean values for amine oxidase activities are quite close considering the ranges involved. Cruz Coke, et al. (16), have reported no difference in the oxygen consumption of normal and ischemic rat kidneys, in their case, however, without a substrate. The greatest apparent change observed in the Dresent study was in the DOPA decarboxylase activity. If the effects on the ratios and presumably on the pressor action are real, they appear to be related to the relative increase in decarboxylase activity. The antihypertensive action of dimercaprol may be similar to the action of hydralazine which reduces DOPA-decarboxylase activity (17). Both will chelate trace metals required for enzymatic activity.
1. Dimercaprol was administered to normotensive and hypertensive rats and their kidney enzyme extracts tested for amino acid decarboxylase and oxidase activities using DOPA as a substrate. Untreated normotensive and hypertensive controls were similarly tested. 2. Although all results were not significant, t h e evidence was suggestive t h a t both the antihypertensive effect of dimercaprol for hypertensive animals a n d i t s pressor action in normotensive animals are related to amino acid metabolism.
REFERENCES (1) Schroeder, H. A,. J. Clin. Invest., 30, 672(1951). (2) Raska, S. B., J . Ezpll. Med., 78,75(1943). (3) Holtz. P.. Heise, R., and Liidtke, K.. Arch. cxpfl. Path. Pharmakol.. 191, 87(1939). (4) Holtz, P., Credner, K., and Koepp, W.. ibid., 200, 356(1942). (5) B‘ing, R. J., A m . J . Physiol., 132,497(1941). 22.5(1041) (6) Bing, R. J., and Zucker, M. B., J . E r p f l . Med., 74. ..,. (7) Oster, K. A,, and Sorkin. S. Z., Proc. SOL.Exptl. Biol. Med., 51, 67(1942). (8) Wolf, H. J., and Heinsen, H. A., Arch. expfl. Pafhol. Pharmakol.. 179, 15(1935). (9) Govaerts, P.. Bull. acad. roy. mLd. B d g . , 4, 357(1939). (10) Schryder, H. A., “Hypertensive Diseases, Causes and Control, Lea and Fehiger, Philadelphia, 1953, p. 133. (11) Page, E. W.. and Reed, R., A m . J . Physiol., 143, ___\.I
__,.
133(194.5~ ___,_”
(12) Drury, D. R., J . Expll. Med., 68, 693(1938). (13) Friedman, M.. and Freed, S. C., Proc. SOC. Exfd. Biol. Med., 70, 670(1949). (14) Schales, O., in “Methods in Enzymology,” Vol. 11. Colowick, S. P., and Kaplan, N. O., Editors, Academic Press Inc., New York. 1855, p. 195. (15) Trelease. S. F., “The Scientific Paper,” 2nd ed., The Williams and Wilkins Company, Baltimore, 1951, p. 26. (16),Cruz Coke, E., Niemeyer, H., and FernandezPo elaire J. Bol. SOC. b i d . s p n . 2, 15(1944). 8 7 ) Pi&, H. M., Jr.. Te;tlebaum, S.. and Schwarti, P. L., Federalion Proc., 14, Part 1, 113(1955).